The human herpesviruses cause a spectrum of clinically significant acute and life-long latent infections and transform cells morphologically and ocogenically. Viral DNA replication is pivotal in the life-cycles of these viruses in that the onset of viral DNA synthesis signals the commitment of infected cells to lysis, whereas the absence of viral DNA synthesis is a hallmark of viability in latently infected and transformed cells. Furthermore, recent evidence has implicated viral DNA replication proteins in generalized recombination, -- a process which may underly the alterations in cellular DNA characteristic of some transformed cells. Using herpes simplex virus type 1 (HSV-1) as a model system, the objectives of this proposal are to elucidate the functions of the seven essential HSV-1 DNA replication proteins in origin-dependent DNA synthesis and generalized recombination, and to investigate the roles of these proteins and specific origin-associated cis-acting elements in the initiation of viral DNA synthesis. For this purpose nonsense, deletion and temperature-sensitive mutations will be generated in cloned copies of the seven HSV-1 genes required for ori-dependent DNA replication. The mutations will then be introduced into the viral genome and the resulting mutants used to assess the involvement of the seven proteins and of their specific intramolecular functional domains in ori-dependent DNA replication, protein-protein interactions, DNA-protein interactions and generalized recombination. Efforts will also be made to identify DNA sequences in HSV-1 ori S required for efficient initiation of ori- dependent DNA synthesis. A better understanding of the mechanisms involved in viral DNA replication should shed light on the disease-producing and transforming properties of herpesviruses and may ultimately provide novel approaches to intervention in the herpesvirus life-cycle.
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